Comprehensive characterization of the embryonic factor LEUTX.

The paired-like homeobox transcription factor LEUTX is expressed in human preimplantation embryos between the 4- and 8-cell stages, and then silenced in somatic tissues. To characterize the function of LEUTX, we performed a multiomic characterization of LEUTX using two proteomics methods and three genome-wide sequencing approaches. Our results show that LEUTX stably interacts with the EP300 and CBP histone acetyltransferases through its 9 amino acid transactivation domain (9aaTAD), as mutation of this domain abolishes the interactions. LEUTX targets genomic cis-regulatory sequences that overlap with repetitive elements, and through these elements it is suggested to regulate the expression of its downstream genes. We find LEUTX to be a transcriptional activator, upregulating several genes linked to preimplantation development as well as 8-cell-like markers, such as DPPA3 and ZNF280A. Our results support a role for LEUTX in preimplantation development as an enhancer binding protein and as a potent transcriptional activator.

Tracking and characterization of partial and full epithelial-mesenchymal transition cells in a mouse model of metastatic breast cancer.

The various stages of epithelial-mesenchymal transition (EMT) generate phenotypically heterogeneous populations of cells. Here, we detail a dual recombinase lineage tracing system using a transgenic mouse model of metastatic breast cancer to trace and characterize breast cancer cells at different EMT stages. We describe analytical steps to label cancer cells at an early partial or a late full EMT state, followed by tracking their behavior in tumor slice cultures. We then characterize their transcriptome by five-cell RNA sequencing. For complete details on the use and execution of this protocol, please refer to Luond et al. (2021).

DUX4 is a multifunctional factor priming human embryonic genome activation.

Double homeobox 4 (DUX4) is expressed at the early pre-implantation stage in human embryos. Here we show that induced human DUX4 expression substantially alters the chromatin accessibility of non-coding DNA and activates thousands of newly identified transcribed enhancer-like regions, preferentially located within ERVL-MaLR repeat elements. CRISPR activation of transcribed enhancers by C-terminal DUX4 motifs results in the increased expression of target embryonic genome activation (EGA) genes ZSCAN4 and KHDC1P1. We show that DUX4 is markedly enriched in human zygotes, followed by intense nuclear DUX4 localization preceding and coinciding with minor EGA. DUX4 knockdown in human zygotes led to changes in the EGA transcriptome but did not terminate the embryos. We also show that the DUX4 protein interacts with the Mediator complex via the C-terminal KIX binding motif. Our findings contribute to the understanding of DUX4 as a regulator of the non-coding genome.

Distinct contributions of partial and full EMT to breast cancer malignancy.

Epithelial-mesenchymal transition (EMT) is a transient, reversible process of cell de-differentiation where cancer cells transit between various stages of an EMT continuum, including epithelial, partial EMT, and mesenchymal cell states. We have employed Tamoxifen-inducible dual recombinase lineage tracing systems combined with live imaging and 5-cell RNA sequencing to track cancer cells undergoing partial or full EMT in the MMTV-PyMT mouse model of metastatic breast cancer. In primary tumors, cancer cells infrequently undergo EMT and mostly transition between epithelial and partial EMT states but rarely reach full EMT. Cells undergoing partial EMT contribute to lung metastasis and chemoresistance, whereas full EMT cells mostly retain a mesenchymal phenotype and fail to colonize the lungs. However, full EMT cancer cells are enriched in recurrent tumors upon chemotherapy. Hence, cancer cells in various stages of the EMT continuum differentially contribute to hallmarks of breast cancer malignancy, such as tumor invasion, metastasis, and chemoresistance.

The long non-coding RNA ET-20 mediates EMT by impairing desmosomes in breast cancer cells.

The vast majority of breast cancer-associated deaths are due to metastatic spread of cancer cells, a process aided by epithelial-to-mesenchymal transition (EMT). Mounting evidence has indicated that long non-coding RNAs (lncRNAs) also contribute to tumor progression. We report the identification of 114 novel lncRNAs that change their expression during TGFß-induced EMT in murine breast cancer cells (referred to as EMT-associated transcripts; ETs). Of these, the ET-20 gene localizes in antisense orientation within the tenascin C (Tnc) gene locus. TNC is an extracellular matrix protein that is critical for EMT and metastasis formation. Both ET-20 and Tnc are regulated by the EMT master transcription factor Sox4. Notably, ablation of ET-20 lncRNA effectively blocks Tnc expression and with it EMT. Mechanistically, ET-20 interacts with desmosomal proteins, thereby impairing epithelial desmosomes and promoting EMT. A short transcript variant of ET-20 is shown to be upregulated in invasive human breast cancer cell lines, where it also promotes EMT. Targeting ET-20 appears to be a therapeutically attractive lead to restrain EMT and breast cancer metastasis in addition to its potential utility as a biomarker for invasive breast cancer.

Phylogenetic and mutational analyses of human LEUTX, a homeobox gene implicated in embryogenesis.

Recently, human PAIRED-LIKE homeobox transcription factor (TF) genes were discovered whose expression is limited to the period of embryo genome activation up to the 8-cell stage. One of these TFs is LEUTX, but its importance for human embryogenesis is still subject to debate. We confirmed that human LEUTX acts as a TAATCC-targeting transcriptional activator, like other K50-type PAIRED-LIKE TFs. Phylogenetic comparisons revealed that Leutx proteins are conserved across Placentalia and comprise two conserved domains, the homeodomain, and a Leutx-specific domain containing putative transcriptional activation motifs (9aaTAD). Examination of human genotype resources revealed 116 allelic variants in LEUTX. Twenty-four variants potentially affect function, but they occur only heterozygously at low frequency. One variant affects a DNA-specificity determining residue, mutationally reachable by a one-base transition. In vitro and in silico experiments showed that this LEUTX mutation (alanine to valine at position 54 in the homeodomain) results in a transactivational loss-of-function to a minimal TAATCC-containing promoter and a 36 bp motif enriched in genes involved in embryo genome activation. A compensatory change in residue 47 restores function. The results support the notion that human LEUTX functions as a transcriptional activator important for human embryogenesis.

An optimised phylogenetic method sheds more light on the main branching events of rhodopsin-like superfamily.

The comparative genomics between different rhodopsin-like family groups (α, ß, γ and δ) is not well studied. We used a combination of phylogenetic analysis and statistical genomic methods to compare rhodopsin-like family proteins in species likely symbolic of this family's evolutionary progression. For intra-cluster relationships, we applied mathematical optimisation to enhance the tree search produced by the neighbour joining method (NJ) and compared it with maximum likelihood (ML) method. To infer inter-clusters relationships, we used Needleman-Wunsch analysis (NW), HHsearch, ancestral sequence reconstruction and phylogenetic network analysis. Using this workflow, we were able to identify key evolutionary events in the rhodopsin-like family receptors. We found that α rhodopsin-like group gave rise to the ß group, while the γ rhodopsin-like group diverged from the ß group. We tracked the diversification of every cluster, revealing that fungal opsin is the most ancient member of the α group, while adenosine receptors could be the first member to diverge in the MECA (melanocortin, endothelial differentiation sphingolipid, cannabinoid, and adenosine receptors) subfamily and that histamine receptors could be the parent of the amines receptors, while hypocretin receptors might be the most ancient member of the ß group. SOG (somatostatin, opioid, galanin) receptors formed the most ancient members of the γ group. Our analysis indicated that basal receptors might be playing a role in early evolution of the nervous system. This is evident in Trichoplax adhaerens genome, where we located histamine receptors and adenosine receptors.

The human PRD-like homeobox gene LEUTX has a central role in embryo genome activation.

Leucine twenty homeobox (LEUTX) is a paired (PRD)-like homeobox gene that is expressed almost exclusively in human embryos during preimplantation development. We previously identified a novel transcription start site for the predicted human LEUTX gene based on the transcriptional analysis of human preimplantation embryos. The novel variant encodes a protein with a complete homeodomain. Here, we provide a detailed description of the molecular cloning of the complete homeodomain-containing LEUTX Using a human embryonic stem cell overexpression model we show that the complete homeodomain isoform is functional and sufficient to activate the transcription of a large proportion of the genes that are upregulated in human embryo genome activation (EGA), whereas the previously predicted partial homeodomain isoform is largely inactive. Another PRD-like transcription factor, DPRX, is then upregulated as a powerful repressor of transcription. We propose a two-stage model of human EGA in which LEUTX acts as a transcriptional activator at the 4-cell stage, and DPRX as a balancing repressor at the 8-cell stage. We conclude that LEUTX is a candidate regulator of human EGA.

Characterization and target genes of nine human PRD-like homeobox domain genes expressed exclusively in early embryos.

PAIRED (PRD)-like homeobox genes belong to a class of predicted transcription factor genes. Several of these PRD-like homeobox genes have been predicted in silico from genomic sequence but until recently had no evidence of transcript expression. We found recently that nine PRD-like homeobox genes, ARGFX, CPHX1, CPHX2, DPRX, DUXA, DUXB, NOBOX, TPRX1 and TPRX2, were expressed in human preimplantation embryos. In the current study we characterized these PRD-like homeobox genes in depth and studied their functions as transcription factors. We cloned multiple transcript variants from human embryos and showed that the expression of these genes is specific to embryos and pluripotent stem cells. Overexpression of the genes in human embryonic stem cells confirmed their roles as transcription factors as either activators (CPHX1, CPHX2, ARGFX) or repressors (DPRX, DUXA, TPRX2) with distinct targets that could be explained by the amino acid sequence in homeodomain. Some PRD-like homeodomain transcription factors had high concordance of target genes and showed enrichment for both developmentally important gene sets and a 36 bp DNA recognition motif implicated in Embryo Genome Activation (EGA). Our data implicate a role for these previously uncharacterized PRD-like homeodomain proteins in the regulation of human embryo genome activation and preimplantation embryo development.

Homeodomain proteins: an update.

Here, we provide an update of our review on homeobox genes that we wrote together with Walter Gehring in 1994. Since then, comprehensive surveys of homeobox genes have become possible due to genome sequencing projects. Using the 103 Drosophila homeobox genes as example, we present an updated classification. In animals, there are 16 major classes, ANTP, PRD, PRD-LIKE, POU, HNF, CUT (with four subclasses: ONECUT, CUX, SATB, and CMP), LIM, ZF, CERS, PROS, SIX/SO, plus the TALE superclass with the classes IRO, MKX, TGIF, PBC, and MEIS. In plants, there are 11 major classes, i.e., HD-ZIP (with four subclasses: I to IV), WOX, NDX, PHD, PLINC, LD, DDT, SAWADEE, PINTOX, and the two TALE classes KNOX and BEL. Most of these classes encode additional domains apart from the homeodomain. Numerous insights have been obtained in the last two decades into how homeodomain proteins bind to DNA and increase their specificity by interacting with other proteins to regulate cell- and tissue-specific gene expression. Not only protein-DNA base pair contacts are important for proper target selection; recent experiments also reveal that the shape of the DNA plays a role in specificity. Using selected examples, we highlight different mechanisms of homeodomain protein-DNA interaction. The PRD class of homeobox genes was of special interest to Walter Gehring in the last two decades. The PRD class comprises six families in Bilateria, and tinkers with four different motifs, i.e., the PAIRED domain, the Groucho-interacting motif EH1 (aka Octapeptide or TN), the homeodomain, and the OAR motif. Homologs of the co-repressor protein Groucho are also present in plants (TOPLESS), where they have been shown to interact with small amphipathic motives (EAR), and in yeast (TUP1), where we find an EH1-like motif in MATα2.

Novel PRD-like homeodomain transcription factors and retrotransposon elements in early human development.

Transcriptional program that drives human preimplantation development is largely unknown. Here, by using single-cell RNA sequencing of 348 oocytes, zygotes and single blastomeres from 2- to 3-day-old embryos, we provide a detailed analysis of the human preimplantation transcriptome. By quantifying transcript far 5'-ends (TFEs), we include in our analysis transcripts that derive from alternative promoters. We show that 32 and 129 genes are transcribed during the transition from oocyte to four-cell stage and from four- to eight-cell stage, respectively. A number of identified transcripts originates from previously unannotated genes that include the PRD-like homeobox genes ARGFX, CPHX1, CPHX2, DPRX, DUXA, DUXB and LEUTX. Employing de novo promoter motif extraction on sequences surrounding TFEs, we identify significantly enriched gene regulatory motifs that often overlap with Alu elements. Our high-resolution analysis of the human transcriptome during preimplantation development may have important implications on future studies of human pluripotent stem cells and cell reprograming.

The Homeobox Genes of Caenorhabditis elegans and Insights into Their Spatio-Temporal Expression Dynamics during Embryogenesis.

Homeobox genes play crucial roles for the development of multicellular eukaryotes. We have generated a revised list of all homeobox genes for Caenorhabditis elegans and provide a nomenclature for the previously unnamed ones. We show that, out of 103 homeobox genes, 70 are co-orthologous to human homeobox genes. 14 are highly divergent, lacking an obvious ortholog even in other Caenorhabditis species. One of these homeobox genes encodes 12 homeodomains, while three other highly divergent homeobox genes encode a novel type of double homeodomain, termed HOCHOB. To understand how transcription factors regulate cell fate during development, precise spatio-temporal expression data need to be obtained. Using a new imaging framework that we developed, Endrov, we have generated spatio-temporal expression profiles during embryogenesis of over 60 homeobox genes, as well as a number of other developmental control genes using GFP reporters. We used dynamic feedback during recording to automatically adjust the camera exposure time in order to increase the dynamic range beyond the limitations of the camera. We have applied the new framework to examine homeobox gene expression patterns and provide an analysis of these patterns. The methods we developed to analyze and quantify expression data are not only suitable for C. elegans, but can be applied to other model systems or even to tissue culture systems.

Identification of novel non-coding RNA-based negative feedback regulating the expression of the oncogenic transcription factor GLI1.

Non-coding RNAs are a complex class of nucleic acids, with growing evidence supporting regulatory roles in gene expression. Here we identify a non-coding RNA located head-to-head with the gene encoding the Glioma-associated oncogene 1 (GLI1), a transcriptional effector of multiple cancer-associated signaling pathways. The expression of this three-exon GLI1 antisense (GLI1AS) RNA in cancer cells was concordant with GLI1 levels. siRNAs knockdown of GLI1AS up-regulated GLI1 and increased cellular proliferation and tumor growth in a xenograft model system. Conversely, GLI1AS overexpression decreased the levels of GLI1, its target genes PTCH1 and PTCH2, and cellular proliferation. Additionally, we demonstrate that GLI1 knockdown reduced GLI1AS, while GLI1 overexpression increased GLI1AS, supporting the role of GLI1AS as a target gene of the GLI1 transcription factor. Activation of TGFß and Hedgehog signaling, two known regulators of GLI1 expression, conferred a concordant up-regulation of GLI1 and GLI1AS in cancer cells. Finally, analysis of the mechanism underlying the interplay between GLI1 and GLI1AS indicates that the non-coding RNA elicits a local alteration of chromatin structure by increasing the silencing mark H3K27me3 and decreasing the recruitment of RNA polymerase II to this locus. Taken together, the data demonstrate the existence of a novel non-coding RNA-based negative feedback loop controlling GLI1 levels, thus expanding the repertoire of mechanisms regulating the expression of this oncogenic transcription factor.

par-1, atypical pkc, and PP2A/B55 sur-6 are implicated in the regulation of exocyst-mediated membrane trafficking in Caenorhabditis elegans.

The exocyst is a conserved protein complex that is involved in tethering secretory vesicles to the plasma membrane and regulating cell polarity. Despite a large body of work, little is known how exocyst function is controlled. To identify regulators for exocyst function, we performed a targeted RNA interference (RNAi) screen in Caenorhabditis elegans to uncover kinases and phosphatases that genetically interact with the exocyst. We identified seven kinase and seven phosphatase genes that display enhanced phenotypes when combined with hypomorphic alleles of exoc-7 (exo70), exoc-8 (exo84), or an exoc-7;exoc-8 double mutant. We show that in line with its reported role in exocytotic membrane trafficking, a defective exoc-8 caused accumulation of exocytotic soluble NSF attachment protein receptor (SNARE) proteins in both intestinal and neuronal cells in C. elegans. Down-regulation of the phosphatase protein phosphatase 2A (PP2A) phosphatase regulatory subunit sur-6/B55 gene resulted in accumulation of exocytic SNARE proteins SNB-1 and SNAP-29 in wild-type and in exoc-8 mutant animals. In contrast, RNAi of the kinase par-1 caused reduced intracellular green fluorescent protein signal for the same proteins. Double RNAi experiments for par-1, pkc-3, and sur-6/B55 in C. elegans suggest a possible cooperation and involvement in postembryo lethality, developmental timing, as well as SNARE protein trafficking. Functional analysis of the homologous kinases and phosphatases in Drosophila median neurosecretory cells showed that atypical protein kinase C kinase and phosphatase PP2A regulate exocyst-dependent, insulin-like peptide secretion. Collectively, these results characterize kinases and phosphatases implicated in the regulation of exocyst function, and suggest the possibility for interplay between the par-1 and pkc-3 kinases and the PP2A phosphatase regulatory subunit sur-6 in this process.

Distinct roles of the Gcn5 histone acetyltransferase revealed during transient stress-induced reprogramming of the genome.

BACKGROUND: Gcn5 belongs to a family of histone acetyltransferases (HATs) that regulate protein function by acetylation. Gcn5 plays several different roles in gene transcription throughout the genome but their characterisation by classical mutation approaches is hampered by the high degree of apparent functional redundancy between HAT proteins. RESULTS: Here we utilise the reduced redundancy associated with the transiently high levels of genomic reprogramming during stress adaptation as a complementary approach to understand the functions of redundant protein families like HATs. We show genome-wide evidence for two functionally distinct roles of Gcn5. First, Gcn5 transiently re-localises to the ORFs of long genes during stress adaptation. Taken together with earlier mechanistic studies, our data suggests that Gcn5 plays a genome- wide role in specifically increasing the transcriptional elongation of long genes, thus increasing the production efficiency of complete long transcripts. Second, we suggest that Gcn5 transiently interacts with histones close to the transcription start site of the many genes that it activates during stress adaptation by acetylation of histone H3K18, leading to histone depletion, probably as a result of nucleosome loss as has been described previously. CONCLUSIONS: We show that stress adaptation can be used to elucidate the functions of otherwise redundant proteins, like Gcn5, in gene transcription. Further, we show that normalization of chromatin-associated protein levels in ChIP experiments in relation to the histone levels may provide a useful complement to standard approaches. In the present study analysis of data in this way provides an alternative explanation for previously indicated repressive role of Gcn5 in gene transcription.

The LIM homeobox gene ceh-14 is required for phasmid function and neurite outgrowth.

Transcription factors play key roles in cell fate specification and cell differentiation. Previously, we showed that the LIM homeodomain factor CEH-14 is expressed in the AFD neurons where it is required for thermotaxis behavior in Caenorhabditis elegans. Here, we show that ceh-14 is expressed in the phasmid sensory neurons, PHA and PHB, a number of neurons in the tail, i.e., PHC, DVC, PVC, PVN, PVQ, PVT, PVW and PVR, as well as the touch neurons. Analysis of the promoter region shows that important regulatory elements for the expression in most neurons reside from -4kb to -1.65kb upstream of the start codon. Further, within the first introns are elements for expression in the hypodermis. Phylogenetic footprinting revealed numerous conserved motifs in these regions. In addition to the existing deletion mutation ceh-14(ch3), we isolated a new allele, ceh-14(ch2), in which only one LIM domain is disrupted. The latter mutant allele is partially defective for thermosensation. Analysis of both mutant alleles showed that they are defective in phasmid dye-filling. However, the cell body, dendritic outgrowth and ciliated endings of PHA and PHB appear normal, indicating that ceh-14 is not required for growth. The loss of a LIM domain in the ceh-14(ch2) allele causes a partial loss-of-function phenotype. Examination of the neurites of ALA and tail neurons using a ceh-14::GFP reporter shows abnormal axonal outgrowth and pathfinding.

Active transcriptomic and proteomic reprogramming in the C. elegans nucleotide excision repair mutant xpa-1.

Transcription-blocking oxidative DNA damage is believed to contribute to aging and to underlie activation of oxidative stress responses and down-regulation of insulin-like signaling (ILS) in Nucleotide Excision Repair (NER) deficient mice. Here, we present the first quantitative proteomic description of the Caenorhabditis elegans NER-defective xpa-1 mutant and compare the proteome and transcriptome signatures. Both methods indicated activation of oxidative stress responses, which was substantiated biochemically by a bioenergetic shift involving increased steady-state reactive oxygen species (ROS) and Adenosine triphosphate (ATP) levels. We identify the lesion-detection enzymes of Base Excision Repair (NTH-1) and global genome NER (XPC-1 and DDB-1) as upstream requirements for transcriptomic reprogramming as RNA-interference mediated depletion of these enzymes prevented up-regulation of genes over-expressed in the xpa-1 mutant. The transcription factors SKN-1 and SLR-2, but not DAF-16, were identified as effectors of reprogramming. As shown in human XPA cells, the levels of transcription-blocking 8,5'-cyclo-2'-deoxyadenosine lesions were reduced in the xpa-1 mutant compared to the wild type. Hence, accumulation of cyclopurines is unlikely to be sufficient for reprogramming. Instead, our data support a model where the lesion-detection enzymes NTH-1, XPC-1 and DDB-1 play active roles to generate a genomic stress signal sufficiently strong to result in transcriptomic reprogramming in the xpa-1 mutant.

Finding ciliary genes: a computational approach.

In the nematode worm Caenorhabditis elegans and several other animal species, many ciliary genes are regulated by RFX (Regulatory Factor binding to the X-box) transcription factors (TFs), which bind to X-box promoter motifs and thereby directly activate ciliary gene expression. This setup (RFX TF/X-box/ciliary gene) makes it possible to search for novel ciliary gene candidates genome-wide by using the X-box promoter motif as a search parameter. We present a computational approach that (i) identifies and extracts from whole genomes genes and the corresponding promoter sequences and annotations; (ii) searches through promoters for regulatory sequence elements (like promoter motifs) by using training sets of known instances of these elements; (iii) scores (evaluates) and sorts all positive hits in a database; and (iv) outputs a list of candidate genes and promoters with a given regulatory sequence element. Evolutionary conservation across species (orthology) of genes, promoters, or regulatory sequence elements is used as an important strengthening feature during the overall search approach. Our computational approach is set up in a modular fashion: not every part needs to be used for a particular search effort. In principle, our approach has broad applications. It applies to any group of genes that share common (conserved) regulation through common (conserved) regulatory sequence elements.